the K , value obtained by solvent extraction. Inasmuch as the ieutral p-PAS has little or no tendenc,y t o form chelates whereas the anionic ??-PAXforms a zinc complex of high stability, it is likely that the quinoid form (c) of the p-PAN anion is probably the best representation of the resonance hybrid, or else is the form stabilized by chelation with the metal ion.
Table V.
Equilibrium Constants for Metal Chelates of 4-(2-Pyridylazo)-l -naphthol
Log KDR 4.2 4.2 4.2 4.2
Metal ion Sone Cu(I1) Zn(I1) Ni(I1)
LOP
KDX
...
3 . 6 =t0 . 2 4.0 =k 0.2
...
Log k'f Solvent extraction ... ... 19.4
...
Log K , Spectrophotometric in 50';7, v./v. aqueous dioxane .. 20 19 23
(2) Chichibabin, A. E.,J . Rum. Phys. Chem. SOC.50, 512 (1920). 13) Corsini, A., Mai-Ling Yih, I., Fernando, &., Freiser. H.; ANAL.CHEW 34,1090 (1962). (4) Corsini, A., Fernando Q., Freiser, H.. Inora. Chern., 2, 224 (1963). (5) Job, P , ,4nn. Chztn. 6, 113 (1936). This compound represents an unusual type of chelation since the protons lost on chelation do not originate in the chelating groups themselves and because the chelating groups (the azo group or the pyridyl nitrogen) are activated only upon the loss of these protons. The effect of metsl chelation uiion p-PAN is the strengthening of 'the acidity of the proton in the p-hydroxy groull ( 4 ) . It odd be interesting in this connection to examine the behavior
of the p-amino analog of this reagent to determine whetner metal chelation would induce the formation of a n imino group in the chelate molecule. An investigation of other chelating agents of this general type, such as 2,4'-dihydroxyazobenzene, is in progress. LITERATURE CITED
D.,Fernando, &., Freiser, H., ANAL.CHEM.35, 294 (1963).
(1) Betteridge,
(6)vent Extraction G. H., in Analytical H .Chem~ istry,>,Kiley, xew york, 1957. ( 7 ) Pollard, F. H,, H ~ p,, G~ ~~ 11. J., A n d . C'hzm. Acta. 20, 26 (1959). ( 6 ) Vosburgh, W C., Cooper, G. n., ?*Torrisonj
Freiserj
(9)Chem. Yoe, J. H., 6 3 ,Jones, 437 (lg41). A L.,I V D .ESG. CHEW,ANAL.En 16, 111 (19k1).
RECEIVED for revieF October 32, 1962. Accepted 1Iarch 7, 1963. 1Vork suppqrted by the U. S. Atomic Energy Cornmission.
A New Color Reaction of Anthrone with Malonaldehyde and Other Aliphatic Aldehydes TAI-WAN K W O N and BETTY M. W A l l S Deparfmenf o f Food crnd Nutrifion, Florida Stafe University, Tallahassee, Fla.
b Anthrone reagent (13.2% anthrone in concentrated sulfuric acid) reacts with a series of short-chain aliphatic aldehydes producing orange to violet-red colors. Absorption curves of the reaction products are given in the visible region. The anthrone complexes of saturated aldehydes (acetaidehyde, butyraldehyde, and hexaldehyde) show their A,, at 476 mp, formaldehyde at 488 mp, and propionaldehyde at 455 mp, with molar absorptivities ranging from 3.0 X 1 O2 to 4.5 X lo3. The anthrone complexes of short chain a,@-unsaturated aldehydes (malonaldehyde, acrolein and crotona!dehyde) produce their A,, at 510 mp, with much higher absc'rption (E = approximately 7.9 X lo3). Possible reaction mechanisms a re discussed.
titatively with carbohydrates, producing a stable green color. The color formation, like that of the Molisch reaction, is believed to depend upon the formation of furfuraldehyde from the carbohydrates by the strong sulfuric acid in the reaction mixture. In preliminary experiments, colors ranging from orange to violet-red were observed when aliphatic aldehydes were reacted with the anthrone reagent. The present paper describes this reaction with several short-chain saturated and unsaturated aliphatic aldehydes. I n particular, quantitative aspects of the malonaldehyde-anthrone reaction have been illustrated in detail, and a possible reaction mechanism is proposed. EXPERIMENTAL
The anthrone reagent was prepared b y a slight modification of the method of Morris (8). Anthrone (Eastman Organic Chemicals, Rochester 3, N. Y.) was dissolved in concentrated sulfuric acid (Baker Chemical Co., Phillipsburg, iY.J.) to give a final concentration of 0.2% (w./v.), Reagent.
A
has been used widely for the quantitative jetermination of carbohydrates (3, 8, IO). The reagent, which consists of anthrone dissolved in concentrated sulfuric al:id, reacts quanNTHRONE
Procedure. Three milliliters of anthrone reagent were added to 2 ml. of aldehyde solution in borosilicate glass test tubes (16- x 150-mm.). The reagent was directly poured into the center of the sample tube t o enSure good miuing. AZt least 10 minutes were allowed for color development. Preparation of Aldehydes. For t h e qualitative absorption spectra of the reaction miutures, the aldehydcs mere prepared as folloivs : Formaldtlhyde (Raker) \vas distilled in glaqs. Acetaldehyde and acrolein man), propionaltlehyde, h u t hyde, crotonaldehytle, and hemldehyde (Columbia Organic Chemicals, Columbia, S.C.) n ere used TI ithout further purification. Malonaldehyde was prepared from malonaldehydc bis(diethyl acetal) (Kay-Fries, Chemicals, Xew I'ork 16, S.Y.) by acid hydrolysis. All aldehydes were diluted n ith distilled nater to a n appropriate range of absorbance and the spectia of their reaction products with anthrone %ere recorded against a reagent blank, using a Bausch & Lomb Spectronic 505 recording spectrophotometer. For the determination of molar absorptivity of the reactioii mixtures, all VOL. 35, NO. 6, M A Y 1963
e
733
~ ~ ~
W I
430
\\\
450
470
490
510
530
550
MP
Figure 1 . Visible absorption spectra of anthrone-aldehyde complexes Concentrations varied; given in Table I
molar absorptivities
of the aldehydes, with the excpption of malonaldehyde, were further purified by use of an Xerograph gas chromatograph Model A-90-A. The column was 5 feet by '/,-inch stainless steel, packed with 20% Silicone GE SF-96 on 60/80 firebrick. Helium flow rate mas 30 cc. per minute. Temperatures of column, injector, and detector were 120", 200", and 220' C., respectively. Five microliters were injected in each case and the separated aldehyde corresponding to the main peak n-as collected in 4 ml. of distilled water. The resulting aldehyde solutions were diluted as necessary and their concentrations determined from ultraviolet spectrophotometric data (6), using a Beckman DU spectrophotometer. I n the case of formaldehyde, the purified fraction, separated from the gas chromatograph, gave slightly lom7er molar absorptivity when reacted with anthrone than did the original compound, obtained by distillation. It is suspected that polymerization may have occurred during the separation. The values presented are, therefore, those from the original distillate. Malonaldehyde could be obtained by hydrolysis of the more stable malonaldehyde bis(diethy1 acetal) in dilute aqueous solution. The commercial preparation was redistilled and the fraction boiling a t 211.5' C. was collected. RESULTS AND
Table 1.
Spectral Data on AnthroneAldehyde Complexes
Limit1%
Aldehyde Amax B x 10-3" c0ncn.b Formaldehyde 488 4 5 34 26 4 Acetaldehyde 476 0.83 Propionaldehyde 455 0.80 36 0 Acrolein 510 8 0 35 Malonaldehyde 510 7 9 4 6 Butyraldehyde 476 0 30 120 Crotonaldehyde 510 8 0 4 4 Hexaldehyde 476 0 33 154 a Based on aldehyde concentrations in diluted reaction mixture at AmSx Dilutionfactor = 2l/9. b Micrograms in 2-ml. test solution t o give 0.1 absorbance in 1-em. eel1 at Amax.
of standing a t the reaction temperature of the mixture. Additional heating in a boiling-water bath did not significantly affect the color, nor did holding the reaction mixture a t room temperature 110%). EFFECTOF REAGENT CONCENTRATION.for 24 hours. Reaction Mechanism. The mechaA sulfuric acid concentration of 95% or nism of the color-forming reaction of higher was necessary for maximum malonaldehyde with anthrone is becolor development. Increasing the anlieved to differ from that of its reaction throne content between 0.05% (w./v.) m-ith other test reagents 111-19). By and 0.2% (w./v.) in the reagent provirtue of its reactive methylene group, duced progressively higher optical denanthrone, in strong sulfuric acid, consity values. However, a higher content denses with various organic compounds than 0.2% (w./v.) failed to give a clear (I,@. With a,P-unsaturated aldehydes reaction mixture, probably because of the -i.e., acrolein produced from glycerolcrystallization of anthrone. The conthe condensation product is benzancentration adopted-0.27, (w./v.) anthrone (2,7, 9). This product dimerizes throne in concentrated sulfuric acidto give either violet-red dibenzanthrone was applied throughout the study. or green iso-dibenzanthrone in concenRATIOOF REAGENT TO SAMPLE.Varitrated sulfuric acid (9). ous ratios of the above reagent solution I n the present study, glycerol, to sample solution were examined. The acrolein, malonaldehyde, and crotonalvolume-to-volume ratio needed to obdehyde all gave violet-red pigments tain a clear reaction mixture after coolwith the anthrone reagent and maxiing to room temperature was about 1.4. mum absorption a t 510 mp. Upon On this basis, a ratio of 1.5 was adopted. dilution with water, yellow precipitates This method yields absorbances about formed which could be collected on a 1.3 times higher than those obtained by glass filter and dried. The dried blorris' method (8). EFFECT OF HEATON COLOR PRODUC- material gave a violet-red color upon addition of sulfuric acid (1.5 to 1.0 H20, TION OF REACTION MIXTURE. When by volume). the reagent was added to the sample From the above considerations, it may solution, heat was evolved and the tembe assumed that 8-hydroxyacrolein, the perature reached about 85" C. This enolic form of malonaldehyde (4,6) and heat is a fundamental condition in proacrolein (or glycerol) follow similar reacducing the color. Maximum color detion mechanisms as follows: velopment was reached after 10 minutes
DISCUSSION
Visible Spectra of Anthrone-Aldehyde Complexes. The colored reaction mixtures showed their maximum absorptions in the region of 455 mp to 510 mp (Figure 1). The spectra obtained from the reaction with acrolein and crotona1dehJ.de were similar to that of malonaldehyde, while the spectra of the butyraldehyde and hexaldehyde anthrone complexes were similar
734
to that of acetaldehyde in the range of 450 to 530 mw. As listed in Table I, the saturated aldehydes produced complexes which absorbed a t lower wavelengths and, except for formaldehyde, had much lower absorptivity than those f r o m n,P-unsaturated a l d e h y d e s . Thus, this color reaction may be useful in the estimation of n$-unsaturnted aldehydes. Details of the reaction between malonaldehyde and anthrone are discussed in the following section. Malonaldehyde-Anthrone ReacAND PRECISION OF tion. LINEARITY DATA. A direct proportionality between absorbance and concentration was obtained in the range 0.5 to 3.0 x lo-' mole of malonaldehyde per ml. of sample solution, using a Beckman DU and a 1-cm. cell. The relative standard deviation of the test results from the mean value was 2.8%, and the extreme range was 96.8 to 104.8% of the mean. A linear response was also obtained with the Bausch & Lomb Spectronic 20, using 1-inch test tubes (relative standard deviation 4.5%, range 96.6 to
ANALYTICAL CHEMISTRY
\ /
\ /
dibenzanthrone (Amx 510 mp)
The violet-red color of the dibenzanthrone may be stskilized by interaction with the H2S0,. I t may be hypothesized that HS04- and HoSO4+ contribute a e1ectron:i to the dibenzanthrone. Crotona1dehg.de Fcacts by a similar mechanism (2, 9). By condensation of crotonaldehyde with anthrone, 11methyl-benzanthrone is obtained (9), and dimerization niay be assumed to follow. To test the proposed mechanism, benzanthrone (Calbiochem, Loa Xngeles 63, Calif.) w,s dissolved in concentrated
and crotonaldehyde (Table I). Evidently the Of t’lese ‘Ompounds with anthrone t o give benzanthrone (or its methyl derivative) is stoichiometric and goes to completion under the test conditions.
comoound similar to that of nmlonaldehyde, except that the 12 and 13 positions in benzanthrone are saturated.
aH H
H2
I
2CH3CH2CHO
a2
+2,
\
0
sulfuric acid and further diluted to give suitable concentrations in a sulfuric acid-water mixture identical with that used in the anthront? test. The molar absorptivity of this solution at the A,, of 510 m p was 7.8 X lo3,very close to the values found for the anthrone reaction products with malorlaldehyde, acrolein
colored dimer b,,455 m d
0
Other saturated aldehydes probably condense with anthrone without cyclization. The structures of the resulting pigments are not known, nor is there evidence on which to base an explanation of the differences noted in the activity of formaldehyde as compared to other saturated aldehydes.
LITERATURE CITED
(1) Allen, C. F. H., Overbaugh, S. C., J. AWL.Chem. SOC.57, 1322 (1935). (2) Bally, O., Scholl, R., Ber. 44, 1656 (1911). (3) Carrel, N. Longlep, R. W., Roe, J. H.. J . Biol. Chem. 220, 583 (1956).
v.,
235 (191
